Sequence specific interactions between nucleic acids by Watson-Crick base pairing, or between nucleic acids and proteins proceed by well-defined recognition rules which govern all steps of gene expression. In principle, specific interference with any such event would provide a means to control cellular or viral gene expression. The antisense strategy has been used in a pharmacological manner to block the expression of various genes (for reviews see: (i) Uhlman, E.; Peyman, A. Chem. Rev. 1990, 90, 543. (ii) Stein, C. A.; Cohen, J. A. Cancer Res. 1988, 48, 2659. (iii) Matteucci, M. D.; Bischofberger, N. Annu. Rep. Med. Chem. 1991, 26, 287. (iv) Miller, P. S.; Ts'o, P. O. P. Annu. Rep. Med. Chem. 1988, 23, 295. (v) Neckers, L.; Whitesell, L.; Rosolen, A; Geselowitz, D. A. Critical Revs. Oncogenesis 1992, 3, 175. (vi) Gene Regulation: Biology of Antisense RNA and DNA, Volume 1, Erickson, R. P.; Izant, J. G., Eds.; Raven Press: New York 1992). Antisense oligonucleotides are short single-stranded DNA or RNA fragments whose nucleotide sequence is complementary to a specific sequence within the target mRNA. The antisense oligonucleotide hybridizes to the mRNA which thereby inhibits gene expression by possibly blocking processing, transport, or translation of the sense mRNA. The inhibition of translation observed may also be due to cleavage of the mRNA by ribonuclease H (RNase H), an enzyme found in the nuclei of mammalian cells that is able to hydrolyze the RNA strand of an RNA-DNA hybrid. Endogenous RNase H-like activity may play a role in the specific inhibiting properties of antisense oligonucleotides observed in cultured cells.
Examples of the success of the antisense strategy using oligonucleotides include inhibition of Herpes simplex virus replication (Kulka, M.; Smith, C. C.; Aurelian, L.; Fishelevich, R.; Meade, K.; Miller, P.; Ts'o, P. O. P. Proc. Natl. Acad. Sci. USA 1989, 86, 6868) and blocking viral protein synthesis of HIV-1 (Agrawal, S.; Ikeuchi, T.; Sun, D.; Sarin, P. S.; Konepka, A.; Maizel, T.; Zamecnik, P. C. Proc. Natl. Acad. Sci. USA 1989, 86, 7790). Antisense oligonucleotides have also been shown to inhibit the expression of specific oncogenes in cell culture, such as c-myc (Wickstrom, E. L.; Bacon, T. A.; Gonzalez, A.; Freeman, D. L.; Lyman, G. H.; Wickstrom, E. Proc. Natl. Acad. Sci. USA 1988, 85, 1028) and c-myb (Gewirtz, A. M.; Calabretta, B. Science 1988, 242, 1303).
The goal in the development of antisense oligonucleotides is to inhibit specific gene expression in intact cells. The desired properties of antisense oligonucleotide and oligodeoxynucleotides includes stability against nucleases, membrane permeability and selective inhibition of gene expression. Unmodified phosphodiester antisense oligodeoxynucleotides and antisense RNA have been shown to inhibit translation of targeted mRNA but are susceptible to rapid degradation by nucleases within the cells as well as in mammalian sera. Therefore, much effort has been made to synthesize oligonucleotide analogs with modified internucleotide linkages e.g., phosphorothioate, (Eckstein, F.; Annu. Rev. Biochem. 1985, 54, 367) methylphosphonate (Ts'o, P. O. P.; Miller, P. S.; Aurelian, L.; Blake, K. R.; Murakami, A.; Agris, C.; Blake, K. R.; Lin, S. -B.; Lee, B. L.; Smith, C. C. Ann. N.Y. Acad. Sci. 1988, 507, 220) phosphorodithioate, (Brill, W. K. D.; Tang, J. -Y.; Ma, Y. -X.; Caruthers, M. H. J. Am. Chem. Soc. 1989, 111, 2321) ethylphosphotriester, (Miller, P. S.; Chandrasegaran, S.; Dow, D. L.; Pulford, S. M.; Kan, L. S. Biochemistry 1982, 21, 5468) and phosphoramidate (Froehler, B.; Ng, P.; Matteucci, M. Nucleic Acids Res. 1988, 16, 4831). The majority of the modifications are directed primarily towards the sugar-phosphate backbone and usually involve a minimal change of ligands around the phosphorous atom to prevent distortion in the geometry of the internucleotide bond and thereby maintain fidelity of oligomer binding while enhancing stability and nuclease resistance. There is as yet no universally applicable oligonucleotide structure to serve as an antisense effector. Unmodified phosphodiester oligodeoxynucleotides offer the advantages of good solubility, efficient and stable hybridization and activation of RNase H, but suffer from poor biological stability and poor cellular uptake. Methylphosphonate oligonucleotide analogs are poorly soluble and are unable to direct cleavage of RNA by RNase H. Phosphorothioates are able to survive longer than unmodified oligonucleotides in cells and media due to their nuclease resistance, however, they enter cells more slowly, possibly a result of stronger binding to one or more cell-surface receptors or other proteins (Loke, S. L.; Stein, C. A.; Zhang, X. H.; Mori, K.; Nakanishi, M.; Subasinghe, C.; Cohen, J. S.; Neckers, L. M. Proc. Natl. Acad. Sci. USA 1989, 86, 3474). Phosphorothioates also suffer from the disadvantages of toxicity and non-specific inhibition of protein and DNA synthesis at concentrations which are near those required for sequence-specific effects. Phosphorothioate and methylphosphonate backbone-modified oligodeoxynucleotides exist as diasteromeric mixtures and form less stable hybrids than normal phosphodiester oligonucleotides (Freier, S. M.; Lima, W. F.; Sanghvi, Y. S.; Vickers, T.; Zounes, M.; Cook, P. D.; Ecker, D. J. in Gene Regulation: Biology of Antisense RNA and DNA, Volume 1, pp.95-107; Erikson, R. P.; Izant, J. G., Eds.; Raven Press: New York 1992) (Miller, P. S.; Yano, J.; Yano, E.; Carroll, C.; Jayaraman, K.; Ts'o, P. O. P. Biochemistry 1979, 18, 5134). Chirality may also be important in the case of phosphorothioates in directing RNase H activation of the phosphorothioate oligodeoxynucleotide-RNA heteroduplex. Agrawal has reported that phosphodiester-linked oligodeoxynucleotides are more efficient than the corresponding phosphorothioate analogs with respect to human RNase H activity (Agrawal, S.; Mayrand, S. H.; Zamecnik, P.; Pederson, T. Proc. Natl. Acad. Sci. USA 1990, 87, 1401). The ability to serve as a template for RNase H may have therapeutic value by mediating, or at least enhancing the antisense effect relative to oligonucleotides that are unable to activate RNase H. However the exact role of an RNase H activity in intact cells remains to be ascertained.
The problems arising for example, from chirality, steric hindrance, or hydrophobicity as well as the potential risk of toxicity and antigenicity in vivo, prompted us to consider oligodeoxynucleotides which are constitutional isomers of biological DNA differing only in bond connectivity. One possible approach to modifying an oligonucleotide to generate a constitutional DNA isomer involves the alteration of the sugar moiety. The reversion of the configuration of the 1' carbon atom of the sugar residue results in .alpha.-oligonucleotide analogs (Morvan, F.; Rayner, B.; Imbach, J. -L.; Chang, D. K.; Lown, J. W. Nucleic Acids Res. 1986, 14, 5019) (Morvan, F.; Rayner, B.; Imbach, J. -L.; Lee, M.; Hartley, J. A.; Chang, D. K.; Lown, J. W. Nucleic Acids Res. 1987, 15, 7027) (Imbach, J. -L.; Rayner, B.; Morvan, F. Nucleosides & Nucleotides 1989, 8, 627). Oligo-.alpha.-deoxynucleotides are nuclease resistant and form stable double helices with complementary DNA or RNA sequences (Gagnor, C.; Bertrand, J. R.; Theret, S.; Lemaitre, M.; Morvan, F.; Rayner, B.; Malvey, C.; Lebleu, B.; Imbach, J. -L.; Paoletti, C. Nucleic Acids Res. 1987, 15, 10419) (Cazenave, C.; Chevrier, M; Thuong, N. T.; Helene, C. Nucleic Acids Res. 1987, 15, 10507). They are capable of antisense inhibition of .beta.-globin mRNA translation via an RNase H independent mechanism (Boiziau, C.; Kurfurst, R.; Cazenave, C.; Roig, V.; Thuong, N. T. Nucleic Acids Res. 1991, 19, 1113). Similarly, Beaucage has recently reported that alternating .alpha.,.beta.-oligothymidylates with alternating (3'-5')- and (5' --5')-internucleotide phosphodiester linkages exhibit enhanced nuclease resistance and hybridize with satisfactory affinity to complementary DNA and RNA (Koga, M.; Moore, M. F.; Beaucage, S. L. J. Org. Chem. 1991, 12, 3757).
In some instances substitution of 2'-deoxy-.beta.-D-ribofuranose by an isomeric sugar residue generates an oligodeoxynucleotide that exhibits selective hybridization to DNA and RNA complements. A pentadecanucleotide prepared from 1-.alpha.-D-arabinofuranosylthymine hybridizes with some selectivtity to complementary RNA rather than DNA (Adams, A. D.; Petrie, C. R.; Meyer, R. B. Nucleic Acids Res. 1991, 19, 3647). Another sugar modification which generates a constitutional DNA isomer is the replacement of the 2'-deoxy-D-ribose backbone by 2'-deoxy-L-erythro-pentose to give enantio-DNA. Enantio-DNA (L-dA.sub.6) has been shown to be resistant to bovine spleen phosphodiesterase and binds complementary RNA preferentially to complementary DNA (Shizuyoshi, F.; Shudo, K. J. Am. Chem. Soc. 1990, 112, 7436).
The 2'-5'internucleotide linkages of oligoadenylates (2'-5')A.sub.n, represent unique examples of naturally occurring constitutional RNA isomers. The (2'-5')A.sub.n oligomers have been detected in a variety of cells and tissues including L1210 cells and human lymphocyctes (Cailla, H.; LeBorne De Kaouel, C.; Roux, D.; Delage, M.; Marti, J. Proc. Natl. Acad. Sci. USA 1982, 79, 4742). The (2'-5')A.sub.n has been suspected to be involved in regulation of cell growth and differentiation and in the antiviral mechanism of interferon (Wells, M.; Mallucci, L. Exp. Cell Res. 1985, 159, 27). In the (2'-5')A pathway interferon and double-stranded RNA activate an enzyme, (2'-5')-oligoadenylate synthetase, to catalyze the formation of oligoadenylates from ATP linked 2'-5' rather than by the usual 3'-5' phosphodiester bonds. The oligoadenylates vary in length from two to fifteen residues. The di-, tri- and tetraadenylates are the most abundant and the amounts of larger oligoadenylates diminish with increasing chain lengths (Samanta, H.; Dougherty, J. P.; Lengyel, P. J. Biol. Chem. 1980, 255, 9807). The (2'-5')A.sub.n subsequently binds and activates an endoribonuclease (RNase L) which is responsible for the nonspecific cleavage of messenger and ribosomal RNAs and thereby inhibits protein synthesis in intact cell systems (Farrell, P. J.; Sen, G. G.; Dubois, M. F.; Ratner, L.; Slattery, R. E.; Lengyel, P. Proc. Natl. Acad. Sci. USA 1978, 75, 5893). Double-stranded RNA is not cleaved during the process (Ratner, L.; Sen, G. C.; Brown, G. E.; Lebleu, B.; Kawakita, M.;Cabrer, B.; Slattery, E.; Lengyel, P. Eur. J. Biochem. 1977, 79, 565).
The biological activity of (2'-5')-oligoadenylates is rapidly lost due to (i) cleavage of the 2'-5' internucleotide bond by a specific 2'-5'-phosphodiesterase which begins from the 2' end and degrades in a processive manner and (ii) one or several phosphatases which dephosphorylate (2'-5')A.sub.n to its core congener. This has led to the synthesis of a plethora of structurally modified (2'-5')A.sub.n analogs designed to improve cellular stability and uptake as well as better characterize its binding and activation of RNase L. For example, the half-life of (2'-5')A.sub.n in tissue culture is three hours; however the replacement of the 3' hydroxyl group of the adenosine moieties of (2'-5')A.sub.n by hydrogen atoms (i.e., cordycepin analogs) retains the properties of achirality and increases the half-life at the internucleotide linkages to seventeen hours against 2'-phosphodiesterase and cellular nuclease activity (Kariko, K.; Reichenbach, N. L.; Suhadolnik, R. J.; Charabula, R.; Pfleiderer, W. Nucleosides & Nucleotides 1987, 6, 497).
The (2'-5')oligo-3'-deoxyadenylates are nontoxic to cells and exhibit a broad spectrum of biological activities (Kariko, K.; Reichenbach, N. L.; Suhadolnik, R. J.; Charubala, R.; Pfleiderer, W. Nucleosides & Nucleotides 1987, 6, 497) (Torrence, P. F.; Imai, L.; Jamoulle, J. C.; Lesiak, K. Chem. Scripta 1986, 26, 191). Cordycepin trimer and its 5'-monophosphorylated analog fail to activate RNase L but do inhibit to some extent HIV-1 reverse transcriptase in vitro with no cell toxicity at a concentration of 62.5 .mu.M (Sawai, H.; Imai, J.; Lesiak, K.; Johnston, M. I.; Torrence, P. F. J. Biol. Chem. 1983, 258, 1671). Furthermore, it appears unlikely that under experimental conditions, the cordycepin trimer serves as a prodrug of cordycepin which has no anti-HIV-1 activity in vitro (Montefiori, D. C.; Sobol, R. W.; Li, S. W.; Reichenbach, N. L.; Suhadolnik, R. J.; Charbula, R.; Pfleiderer, W.; Modliszewski, A.; Robinson, W. E.; Mitchell, W. M. Proc. Natl. Acad. Sci. USA 1989, 86, 7191).
Three adenosine monophosphate residues linked 2'-5' and a 5'-phosphorylated moiety are required for binding RNase L. For activation of RNase L, a 5'-di- or 5'-triphosphate is required (Kariko, K.; Reichenbach, N. L.; Suhadolnik, R. J.; Charubala, R.; Pfleiderer, W. Nucleosides & Nucleotides 1987, 6, 497). When the 2'14 5' phosphodiester bond(s) of a 2'-5'A trimer are replaced with 3'-5' phosphodiester linkages a 10.sup.5 -fold decrease in inhibition of translation and a 13,000-fold decrease in ability to bind to RNase L are observed (Lesiak, K.; Imai, J.; Floyd-Smith, G.; Torrence, P. F. J. Biol. Chem. 1980, 258, 13082). There is no detectable 5'-rephosphorylation of the (2'-5')-3'-dA.sub.n core of trichloroacetic acid (TCA)-soluble cytoplasmic extracts of lymphocytes and lymphoblasts (Suhadolnik, R. J.; Doetsch, P. W.; Devash, Y.; Henderson, E. E.; Charubala, R.; Pfleiderer, W. Nucleosides & Nucleotides 1983, 2, 351).
It is unlikely that long nonphosphorylated (2'-5')-3'-dA.sub.n oligomers (n&gt;4) will bind and activate RNase L or inhibit protein synthesis, (Lee, C.; Suhadolnik, R. J. FEBS Lett. 1983, 1, 205) however, they may have antimitogenic properties in intact cells (Nucleosides & Nucleotides 1983, 2, 351). Furthermore substitution of one the adenosine moieties of a (2'-5')A trimer with uridine results in a marked decrease in binding and activation of RNase L (Kitade, Y.; Alster, D. K.; Pabuccuoglu, A.; Torrence, P. F. Bioorg. Chem. 1991, 19, 283).
Based on these highly defined structural requirements the interaction of (2'-5')oligo-3'-deoxynucleotides with RNase L appears selective for adenosine residues of n&lt;4 bases. Thus, it would not be expected that mixed base sequences of longer oligomers (.apprxeq.21 mers), commonly used as modulators of gene expression, containing 3'-deoxy-(2'-5') internucleotide linkages would non-specifically inhibit protein synthesis by the (2'-5')A system.
In order for a 2'-5' oligonucleotide to serve as an effective analog to inhibit gene expression via an antisense or antigene strategy it must bind with complementary base sequences in the target nucleic acid. Theoretical studies on the stability of helices with 2'-5' linked nucleic acids have led to conflicting predictions (Anukanth, A.; Pannuswamy, P. K. Biopolymers 1986, 25, 729; Srinivasan, A. R.; Olson, W. K. Nucleic Acids Res. 1986, 14, 5461). Conformational analysis of dimer and trimer units of (2'-5')A.sub.n, (n=2,3) by nuclear magnetic resonance and circular dichroism studies indicate that the 2'-5' nucleotides show a stronger tendency to base stack even at elevated temperatures than the corresponding 3'-5' ribonucleotides (Doornbos, J.; Den Hartog, J. A. J.; van Boom, J. H.; Altona, C. Eur. J. Biochem. 1981, 116, 403; Johnston, M. I.; Torrence, P. F. in Interferons, Volume 3, pp.189-298; Friedman, R. M., Ed.; Elsevier: Amsterdam, 1984; Torrence, P. F. in Biological Response Modifiers--New Approaches to Disease Intervention, pp.77-105; Torrence, P. F., Ed. Academic: New York, 1985; Lengyl, P. Annu. Rev. Biochem. 1982, 51, 251). Recently Turner has provided experimental evidence that complementary decamers of 2'-5' linked oligoribonucleotides can form antiparallel duplexes by Watson-Crick hydrogen bonding (Kierzek, R.; He, L.; Turner, D. H. Nucleic Acids Res. 1992, 20, 1685). The overall stability, however, of the 2'-5' duplexes is less than the corresponding 3'-5' duplexes, presumably due to a less favorable enthalpy change for association.
In the 3'-deoxynucleotide series, 2'-5' helices of mixed sequences and homopolymers also weakly strand associate as shown by Tm studies and a mobility shift assay (Dougherty, J. P.; Rizzo, C. J. Breslow, R. J. Am. Chem. Soc. 1992, 114, 6254). The association between complementary (2'-5') oligo-3'-deoxynucleotides was shown to improve when uridine was substituted for thymidine (Hashimoto, H.; Switzer, C. J. Am. Chem. Soc. 1992, 114, 6255). The complementary (2'-5')oligo-3'-deoxynucleotides da.sub.12 and dU.sub.12 exhibit a Tm of 22.8.degree. C. versus 40.8.degree. C. for the (3'-5')-linked DNA helix at high salt (Hashimoto, H.; Switzer, C. J. Am. Chem. Soc. 1992, 114, 6255).
The attractive features of conformational flexibility, high biological stability, low cell toxicity and the natural phosphodiester structure suggests that (2'-5')oligo-3'-deoxynucleotides represent a novel backbone structure to serve as an effective antisense inhibitor of gene expression in mammalian cells. An essential requirement in the antisense approach is that an oligonucleotide or its analog recognize and bind tightly to its complementary RNA sequence. The possibility of a 2'-5' oligomer associating with complementary 3'-5' nucleic acids has not been reported. It is the purpose of this invention to provide 2'-5' oligonucleotides for use in therapies for sequence specific inhibition of gene expression via hybridization to complementary mRNA or complementary duplex DNA.
Novel methodologies to evaluate large numbers of oligonucleotides with therapeutic value have recently been reported (Ellington, A. D.; Szostak, J. W. 1992, Nature, 355, 850) (Tuerk, C.; Gold, L. Science 1990, 249, 505) (Ellington, A. D.; Szostak, J. W. Nature, 1990, 346, 818). An experimental procedure called SELEX (systematic evolution of ligands by exponential enrichment) has been described as a general way to study protein-nucleic acid interactions (Tuerk, C.; Gold, L. Science 1990, 249, 505). In this procedure random pools of oligonucleotides containing approximately 10.sup.13 different molecular species, each having a different nucleotide sequence are synthesized. These pools are then incubated with the target molecule, and substances that bind with the highest affinity are isolated by physical separation techniques, such as affinity chromatography or filter binding. The isolated pool is then amplified by enzymatic procedures, and the binding, selection and amplification cycles are repeated until the pool is enriched with only those oligonucleotides that have the greatest affinity. This technique allows for the selection of oligonucleotides that, by chance, have the correct three-dimensional structure to bind to a target molecule. In subsequent steps, the high-affinity oligonucleotides are evaluated for their ability to inhibit activity, for example, enzymatic activity of the target to which they bind.
Aptamers having an affinity to large proteins or small organic target structures, can be selected. Thus, high-affinity inhibitors can potentially be found for any extracellular target molecule for which a therapeutic benefit may be derived. Most importantly, aptamer selection steps can be manipulated to screen the aptamer pool by more criteria than mere affinity for a given target molecule. Thus, other properties that are essential for therapeutic success can be conferred upon the final oligonucleotide.